Copper serves as a cofactor for many enzymes involved in important biological processes, but can also facilitate the formation of toxic organic and oxygen radicals. A host of proteins, including membrane transporters, metallochaperones, and metalloregulatory proteins, maintains intracellular copper concentrations such that copper ions are provided to essential enzymes, but do not accumulate to deleterious levels. Understanding how these proteins function on the molecular level is the theme of this ongoing research program. Despite significant progress toward determining how soluble copper trafficking proteins bind metal ions, recognize physiological partners, and facilitate metal ion transfer, there are large gaps in the current understanding of copper homeostasis, particularly regarding copper translocation across membranes by P1B- type ATPases. Importantly, mutations in human Cu+transporting P1B-type ATPases lead to Wilson disease and Menkes syndrome, serious disorders of copper metabolism. To complete the molecular picture of copper trafficking and to advance understanding of Cu+ATPases, a model system from the hyperthermophile Archaeoglobus fulgidus is being investigated. The A. fulgidus CopA Cu+ATPase contains all of the structural elements that are present in the human Wilson and Menkes disease proteins, including soluble metal binding domains (MBDs), an ATP binding domain (ATPBD), and an actuator domain (A-domain). Other components of the A. fulgidus pathway include a novel CopZ copper chaperone and a putative transcriptional regulator, CopT. The proposed research involves biophysical and structural characterization of CopA, CopZ, and CopT. The soluble CopA MBDs will be structurally characterized and protein-protein interactions between the MBDs and the CopZ chaperone will be investigated. The copper binding properties, potential interactions with CopZ, and structure of CopT will be probed. Finally, state-of-the-art crystallization techniques for membrane proteins will be applied to CopA, and structures determined in multiple conformations. These data will provide molecular insight into the molecular basis for Wilson and Menkes diseases as well as adding to the database of membrane protein structures. A number of human diseases are linked to deficiencies in cellular handling of copper, which is an essential yet potentially toxic metal ion. This project will provide a molecular picture of the type of protein that is defective in Wilson disease and Menkes syndrome, both genetic disorders of copper metabolism. These same proteins may also be associated with resistance to anticancer drugs.